The single stranded circular DNA (ssDNA) viruses that infect eukaryotic hosts belong to several different virus taxonomic families (Van Regenmortel et al., 1999; Pringle, 1999). Circoviruses, Circinoviruses (Mushahwar et al., 1999), Gyroviruses and Parvoviruses infect vertebrates; some Parvoviruses (subfamily Densovirinae) also infect invertebrate hosts while Geminiviruses and viruses in the genus Nanovirus infect plants. There is recent evidence that the viruses currently classified as Circoviruses evolved from Nanoviruses and have switched from a plant to a vertebrate host (Gibbs and Weiller, 1999).
Geminiviruses, Nanoviruses, and Circoviruses are all small circular ssDNA viruses that appear to be fairly closely related, in that they use the same basic rolling-circle mechanism of replication (RCR) and employ very similar life cycle strategies. Recently published data indicate that some plant RCR viruses—dicot-infecting begomoviruses and at least one Nanovirus genomic component even co-exist in some plant infections, with the geminiviral component of the infection presumably providing movement and propagation functions for the Nanovirus element, which functions as a sort of autonomously replicating satellite virus (Mansoor et al., 1999; Saunders and Stanley, 1999). The genomes of all of the plant RCR viruses, and related vertebrate-infecting Circoviruses are small, single-stranded and circular. The Geminiviruses have mono- or bi-partite genomes, with each genomic component between 2.5 and 3.0-kb. The Nanoviruses have multipartite genomes, generally with at least six, and up to ten, circular subgenomic ssDNAs, each of about 1.0-kb (Katul et al., 1998; Boevink et al., 1995; Burns et al., 1995). The Circoviruses PCV and BFDV have circular ssDNA genomes between 1.75- and 2.0-kb that encode at least two proteins. It is hypothesized that the PCV and BFDV genomes evolved after a recombination event between at least two Nanovirus subgenomic component and a vertebrate RNA-infecting virus which contributed a small portion of the new virus's replication associated protein.
The life cycle of the plant RCR viruses and Circoviruses consists of the following stages, (reviewed by Palmer and Rybicki, 1998; Hanley-Bowdoin et al., 1999):    1. Entry of the ssDNA of the virus into the cytoplasm of the host cell as virion or ssDNA-protein complex.    2. Entry of the ssDNA into the host cell nucleus. This could be a passive process, or may be mediated by the viral capsid protein and/or movement proteins (Lazarowitz, 1999)    3. Conversion of the ssDNA genome into dsDNA presumably mediated by the host DNA repair system. This conversion of the virion DNA into circular dsDNA is required for replication of all RCR replicons, as the “replicative form” (RF) dsDNA intermediate is the template for transcription of the viral genome and therefore expression of viral proteins. The RF DNA becomes associated with host histone proteins and exists as a minichromosome-like structure in the nucleus of infected cells (Abouzid et al., 1988).    4. Transcription of “early” genes—those required for viral replication—by the host RNA polymerase II complex. Production of the viral replication-associated protein (Rep) then results in initiation of RCR of the RF DNA.    5. When the viral RF form reaches a certain critical concentration level in the host cell nucleus, viral transcription regulatory proteins down-regulate transcription of early genes, and stimulate transcription of the viral “late” genes, including the structural protein/s and proteins required for dissemination of the viral genome.    6. The “late” viral proteins sequester ssDNA produced during replication, move it out of the cell nucleus and ultimately out of the infected cell, either as a ssDNA-protein complex, or as assembled virions.
The plant RCR viruses and their relatives the Circoviruses all encode a replication-associated protein (Rep) that is absolutely required for replication of the virus genomic components (Mankertz et al., 1998; Elmer et al., 1988; Hafner et al., 1997). All other proteins are dispensable for replication, and may be involved in such functions as: movement from cell-to-cell; encapsidation of the virus genome; shuttling of the virus genome between the nucleus and the cytoplasm of infected cells; transcriptional activation or repression of genes in the host or viral genome. The Rep proteins of these RCR viruses bear some distant relationship to replication initiator proteins of some ssDNA plasmids, as well as of members of the Microviridae, such as coliphage phiX174 (Ilyina and Koonin, 1992), and has led to speculation that the plant RCR viruses and Circoviruses evolved from prokaryotic ssDNA replicons. The Rep proteins of all of these replicons is a sequence specific DNA binding protein with site specific cleavage and joining activity. In all cases, Rep, probably in association with host enzymes and possibly other viral proteins (Castellano et al., 1999) binds RF DNA at specific sequences and nicks the plus strand at a specific point. In the plant RCR viruses and Circoviruses this specific point occurs within a conserved nonanucleotide sequence that occurs in the loop of a stem-loop structure in the viral intergenic region. The sequence of this nonanucleotide sequence is well conserved between all RCR viruses of plants and Circoviruses: in Geminiviruses the sequence of the nonanucleotide origin of RCR is: TAATATTAC (Palmer and Rybicki, 1998; Hanley-Bowdoin et al., 1999); in Nanoviruses (Refs) and Circoviruses the sequence is TANTATTAC (Meehan et al., 1997; Hamel et al., 1998; Morozov et al., 1998) Thus the consensus sequence for nonanucleotide origin of replication for these viruses is TANTATTAC. The Rep protein-mediated cleavage of this nonanucleotide sequence occurs between positions 7 and 8. The minimum amount of sequences that are required to be present on a DNA molecule so that it can be replicated in a reaction mediated by an RCR virus Rep protein are referred to as the RCR virus's minimal origin of replication (minimal ori). The minimal origin of replication is empirically determined, and virus species-specific; the term “minimal ori” is used interchangeably with “ori”, and “origin of replication”. In general, the minimal ori includes: (1) the viral stem-loop structure with TANTATTAC nonanucleotide sequence present in the loop; (2) generally, at least 90 base pairs 5′ to the start of the stem-loop structure and (3) generally, at least 10, but in many cases up to 100 bases, 3′ of the end of the stem-loop structure. The minimal ori is always contained within the main viral intergenic region. The main viral intergenic region (IR) is a non-coding DNA sequence that contains the stem-loop structure, TANTATTAC sequence, binding sites for the Rep protein, the minimal ori, and promoter sequences for driving transcription of viral genes in both orientations relative to the IR. In Geminiviruses of genus Begomovirus, the minimal ori is contained within the common region, a sequence within the IR that is common to both DNA A and DNA B genetic components since the sequence is required to be in present in cis for replication of both components. Likewise, the minimal ori of Nanoviruses is contained within the viral common region, present on all genome components. In Curtoviruses, the minimal ori is contained within the IR, and Mastreviruses the minimal ori is within the Long IR, but sequences in the Short IR are also required for replication. In Circoviruses the minimal ori is contained within the IR, and constitutes the stem-loop structure, TANTATTAC sequence and sequences flanking the stem-loop structure (Mankertz et al., 1997).
Replication of the plant RCR viruses and Circoviruses is entirely dependent upon a single virally-encoded replication initiator protein (Rep). Rep proteins of these viruses all contain three conserved protein motifs which are also present in replication initiator proteins from prokaryotic RCR replicons (Ilyina and Koonin, 1992; Palmer and Rybicki 1998; Mankertz et al., 1998; Meehan et al., 1997; Bassami et al., 1998; Gibbs and Weiller 1999). The function of motif I (FTLNN (SEQ ID NO:7) in Circoviruses, FTLNY (SEQ ID NO:8) in Nanoviruses and FLTYP (SEQ ID NO:9) in Geminiviruses), is unknown; Motif II (GXXXHLQGF (SEQ ID NO:10) in Circoviruses, GXXHLQGF (SEQ ID NO:11) in Nanoviruses and GXXHLH(A/V)L (SEQ ID NO:12) in Geminiviruses) and is probably involved in metal ion coordination. Motif III [(V/N)(R/K)XYXXK (SEQ ID NO:13) in all three groups] contains a conserved tyrosine residue that participates in phosphodiester bond cleavage and in the covalent linkage of Rep to the 5′ terminus of the nicked nonanucleotide motif at the origin of replication. The Rep proteins of these three groups of viruses also contains a fourth conserved motif, a nucleotide triphosphate-binding domain (GX4GKXXWARX28-29DD) (SEQ ID NO:14) that may indicate that these proteins possess helicase activity.
Apart from their functions in RCR, Rep proteins and ancillary replication-associated “early” gene products also seem to have transcription factor activity, and are capable of controlling viral and perhaps also host gene expression. Geminivirus Rep proteins can interact with both mammalian and plant Retinoblastoma protein (Rb) homologues (Xie et al., 1995; 1996; Grafi et al., 1996; Xie et al., 1996; Ach et al., 1997). Rb belongs to a protein family that controls cell cycle progression by sequestering transcription factors necessary for entry of the cell cycle into S phase. There is also evidence that infection of plants with Geminiviruses such as tomato golden mosaic begomovirus (TGMV) is associated with an increase in the levels of proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor required in cellular DNA replication (Nagar et al., 1995). These viruses thus appear to possess the ability to modify the host environment to one that allows viral DNA replication. At present, the exact mechanisms by which these viruses modify the host cell cycle are unclear. This could be achieved exclusively through interaction of viral proteins (such as Rep) with host proteins (such as Rb). It is also possible that transcriptional activation or repression of host genes mediated by the transcription factor activity of viral protein/s may also be involved in resetting the cellular environment to one that is permissive for viral replication.
Of this group of closely related RCR viruses, only Geminiviruses have been exploited as gene vectors in plant cells. Recombinant viral vectors that have a foreign gene inserted in place of a begomovirus coat protein can sometimes infect permissive dicotyledonous plant hosts and move systemically in infected plants (Ward et al., 1988; Hayes et al., 1988; Sudharsha et al., 1998). Vectors that contain part of the begomoviral genome, including at least three open reading frames (AC1 [=Rep],AC2 and AC3) driven by their own promoters, and containing the viral origin of replication, can replicate in transfected dicotyledonous plant cells Palmer et al., (1997). Mastrevirus-derived vectors that contain the two genes (Rep and RepA) necessary for replication of the viral genome and expression of the viral late genes, together with the viral origins of replication, can replicate in cells derived from monocotyledonous cereal plants (Palmer et al., 1997; Palmer et al., 1999).